Biochemical and immunocytochemical analyses were performed to resolve how glutamate and glutamine are handled in rat cerebellar cortex in acute hypoosmotic stress. Rats were subjected to a 15-20% reduction in plasma osmolality by intraperitoneal injection of distilled water and then perfusion fixed after 4 or 8 h survival. Some rats in the latter group had their plasma isoosmolality restored by injections of hypertonic saline 4 h prior to perfusion. Water loading caused a pronounced increase in the tissue level of glutamine and an equimolar decrease in the level of glutamate after 4 h survival. The increase in glutamine was transient, as judged by analyses at 8 h survival. Light microscopic immunocytochemistry revealed a pronounced enhancement of the glutamine immunolabelling of glial cells (Golgi epithelial cells and astrocytes), including their perivascular end feet, and quantitative immunogold analyses at the electron microscopic level showed that this enhancement reflected a 50% increase in the intracellular concentration of fixed glutamine. Since water loading was associated with glial swelling this change corresponded to a several-fold increase in the glial content of glutamine. There was a modest reduction in the overall staining intensity for glutamate. The biochemical and immunocytochemical changes were reversed upon restoration of plasma osmolality by hypertonic saline. These findings suggest that hypoosmotic stress causes an increased conversion of glutamate to glutamine in glial cells and that the latter amino acid is subsequently lost from the tissue. The flux of glutamate carbon skeletons through the glutamine synthetase pathway in glia, prior to an efflux to the systemic circulation, may explain how glutamate, and excitatory transmitter and potential toxin, can be used as an organic osmolyte in brain tissue.